The present invention relates to a process for the production of D-xcex1-amino acids by the stereospecific conversion of racemic mixtures of 5-substituted hydantoins with a microorganism transformed with a plasmid capable of espressing in high yields and without inducers an enzymatic system capable of directly converting said hydantoins into the corresponding D-xcex1-amino acids.
The term enzymatic system refers to a system consisting of D-hydantoinase and D-N-carbamylase enzymes.
D-xcex1-amino acids are extremely valuable compounds useful for the preparation of pharmacologically active substances (for example, D-phenylglycine and D-para-hydroxyphenylglycine are used in the synthesis of penicillins and cephalosporins), pesticides (D-valine for the synthesis of the insecticide fluvanilate) or sweeteners (D-alanine).
The preparation of D-xcex1-amino acids by the chemical and/or enzymatic hydrolysis of the corresponding 5-substituted hydantoins is known in the art.
For example patent FR 2.310.986 describes a process wherein 5-substituted hydantoins are chemically hydrolized into racemic mixtures of D,L amino acids which are subsequently subjected to a separation treatment of the isomer of interest.
Patent FR 2.317.357, on the contrary, describes a process wherein racemic mixtures of 5-substituted hydantoins are subjected to enzymatic hydrolysis and, subsequently, the products of this transformation (N-carbamyl-D-xcex1-amino acids) are chemically oxidized into the corresponding D-xcex1-amino acids.
The problems relating to these processes generally consist in the fact that they require complex procedures for the resolution and purification of the D-xcex1-amino acids. As a result these processes are not of economical interest from an industrial point of view.
Processes are described in the art wherein D-xcex1-amino acids are obtained directly from 5-substituted hydantoins by the treatment of these with enzymatic systems prepared from microorganisms such as Paeudomonas, Moraxella, Agrobacterium, Hansenula, Arthrobacter (EP-199.943, EP-309.310, U.S. Pat. No. 4,312,948, FR 2456728).
The preparation of these enzymatic systems, however, requires the use of efficient inducers capable of stimulating the production of these enzymes on the part of the microorganisms. It is, in fact, known that the expression level of the enzymes D-hydantoinase and D-N-carbamylase is constitutively very low (Syldatk et al. (1990), xe2x80x9cAdvances in Biochem. Engineering/Biotechnology (Fiechter, A. Ed.), 41, pages 29-75, Springer-Verlag, Berlin).
The inducers normally used are derivatives of hydantoins or nitrogenated cyclic compounds which are howevery easily metabolized by the microorganisms, or compounds such as uracil or thio-2-uracil or thymine which are not metabolized (Meyer et al., (1993), Fems Microbiol. Letters, 109: 67-74).
The use of inducers creates a series of drawbacks among which an increase in the production costs and a certain variability in the production yields of the enzymes. In addition, the expression level which can be obtained in most of the microorganisms following induction is insufficient for economical use in industrial processes (Syldatk et al. (1987), Biotechnol. lett., 9: 25-30; Yokozeki et al. (1987) Agric. Biol. Chem., 51, 715-722).
Recently the genes which encode the enzymes D-hydantoinase and D-N-carbamylase have been individually sequenced and cloned (U.S. Pat. No. 4,912,044 and EP-515-698).
More specifically, patent U.S. Pat. No. 4,912,044 describes the preparation of D-hydantoinase by the fermentation of a microorganism transformed with a hybrid vector containing the hydantoinase gene whose expression is induced by temperature variation. The enzyme thus obtained is used for the production of D-N-carbamyl derivatives from 5-substituted hydantoins.
Patent application EP-515.698 describes, on the other hand, the preparation of D-N-carbamylase by the fermentation of a microorganism transformed with a plasmid comprising the carbamylase gene whose expression is chemically induced with IPTG. The enzyme thus obtained is used for the production of D-xcex1-amino acids from N-carbamyl derivatives.
As industrial interest is directed towards the conversion of racemic hydantoins to D-xcex1-amino acids, the fact that the two enzymes are expressed in different strains involves the use of both and consequently the development of a process starting from two distinct fermentative processes.
This obviously increases the production costs and reduces the conversion kinetics. In fact, in order to complete the enzymatic reaction, the N-carbamyl derivative produced by the transformed microorganism containing the hydantoinase must pass through the bacterial membrane, spread into the reaction medium and then proceed in the opposite direction to reach the second enzyme (carbamylase) present in the other strain. All this is particularly penalizing from the point of view of kinetics considering the reduced permeability of the bacterial membranes to the carbamyl derivatives (Olivieri et al. (1981), Biotechnol. Bioeng., 23, 2173-2183) and the inevitable dilution of the carbamyl itself in the reaction mixture.
Finally, the use of a double volume of biomass has a negative influence on the yields and degree of purity of the final product.
In addition, the necessity of having to induce the expression of these enzymes creates a further problem thus making these processes of little interest for practical use.
The object of the present invention is to overcome the disadvantages of the known art described above.
In particular it has now been found, in accordance with the present invention, that the use of a particular plasmid which contains the genes of D-hydantoinase and N-carbamylase put under the control of an appropriate synthetic promoter, enables the high expression of these enzymes to be obtained without inducers.
It is therefore possible to prepare a single microorganism transformed with said plasmid containing the two enzymatic activities inside. This solution solves not only the problems relating to kinetics due to the limited permeability, as the two reactions occur inside the same cell where the concentration of the substrates is excellent, but also those relating to the requirement of inducers and treatment of the product and of the waste products.
In accordance with this, a first aspect of the present invention relates to a process for the production of D-xcex1-amino acids by the stereospecific conversion of racemic mixtures of 5-substituted hydantoins characterized in that, the conversion reaction is carried out in the presence of a microorganism transformed with a plasmid capable of expressing at high levels and without inducers an enzymatic system capable of converting said hydantoins into the corresponding D-xcex1-amino acids.
A further object of the present invention is the plasmid pSM651 comprising the genes which encode the enzymatic system.
Yet another object of the present invention is a microorganism transfored with the plasmid pSM651 capable of expressing with high efficiency and without inducers an enzymatic system capable of stereospecifically converting racemic mixtures of 5-substituted hydantoins into the corresponding D-xcex1-amino acids.
A further object of the present invention relates to the use of said microorganisms or enzymatic system isolated from said microorganisms for the production of D-xcex1-amino acids by the stereospecific conversion of racemic mixtures of 5-substituted hydantoins.
Further objects of the present invention will be evident from the description and examples below.
FIG. 1: Map of the plasmid pSM637 containing the carbamylase gene
FIG. 2: Map of the plasmid pSM650 containing the hydantoinase gene
FIG. 3: Map of the plasmid pSM651 containing the hydantoinase-carbamylase operon.
FIGS. 4A-B: Nucleotide and amino acid sequence of carbamylase (SEQ ID NO:18-19).
FIGS. 5A-C: Nucleotide and amino acid sequence of hydantoinase (SEQ ID NO:20-21).
FIGS. 6A-B: SDS-PAGE (A) and Western-Blot (B) of the total proteins extracted from cultures of E.coli and B.subtilis transformed with the plasmid pSM651 wherein:
line 1: standard hydantoinase
line 2: standard carbamylase
line 3: E.coli (pSM671) control
line 4: E.coli SMC305
line 5: B.subtilis (pSM671) control
line 6: B.subtilis SMS373
The genes which encode the D-hydantoinase and D-N-carbamylase enzymes can be isolated from microorganisms such as Pseudomonas, Hansenula, Agrobacterium, Aerobacter, Aeromonas, Bacillus, Moraxella, Brevibacterium, Flavobacterium, Serratia, Micrococcus, Arthrobacter or Paracoccus. Specific examples of these microorganisms are Bacillus macroides ATCC 12905, Aerobacter cloacae IAM 1221, Agrobacterium sp. IP I-671, Agrobacterium radiobacter NRRLB 11291, Pseudomonas sp. FERM BP 1900.
The isolation of the genes which encode the D-hydantoinase and D-N-carbamylase enzymes can be carried out by the construction of a gene library, representing the genome of the microorganism, identification of the clones containing the genes which encode said enzymes, analysis of the gene sequence, insertion of said genes into a vector and control of their expression.
The term gene library or genome bank means the combination of clones of a given host microorganism each of which carries a fragment of the chromosomal DNA deriving from the donor organism of which the bank is to be obtained. A bank is defined as being representative when the combination of the single fragments contained in each clone forms the majority of the chromosomal DNA of the donor organism.
According to a preferred embodiment of the process of the present invention, the strain A.radiobacter NRRL B-11291 is used as donor organism for the isolation of the genes which encode D-hydantoinase and D-N-carbamylase.
In practice, two genome banks of said microorganism are constructed in E.coli by digesting the chromosomal DNA separately with the restriction enzymes BamHI and SacI. Among the fragments obtained with the two digestions, those having dimensions normally of between 3,000 and 4,500 bp are purified. The selection is carried out by estimating the molecular weight of the D-hydantoinase and D-N-carbamylase enzymes of 50,000 and 34,000 Daltons respectively.
The two populations of BamHI and SacI fragments are then ligated to a vector of E.coli under such conditions as to facilitate the condensation of a single fragment to each molecule of the vector. The two ligase mixtures are used to transform cells of E.coli made competent as shown for example by Dagert, M. and Ehrlich (1979), (Gene, 6:23).
The two populations of colonies (genome banks) thus obtained, each of which carrying a hybrid plasmid i.e. consisting of the molecule of the vector and a chromosomal DNA fragment of A.radiobacter, are then selected to identify those clones containing the hydantoinase and carbamylase genes.
The identification can be carried out by direct expression or using specific probes. The second method is preferably used. For the selection of the probes, in the case of carbamylase, reference was made to the knowledge of the amino-end sequence of carbamylase by Comomonas sp. 5222c (Ogawa et al. (1993), Eur. J. Biochem., 212: 685-691).
On the basis of this sequence small oligonucleotides are synthesized which, once marked, are used for the screening of the genothecas by hybridization techniques (Maniatis et al., (1982), xe2x80x9cMolecular Cloning: a laboratory manualxe2x80x9d, Cold Spring Harbor Laboratory).
This permitted the identification of a clone carrying a hybrid plasmid carrying a BamHI fragment containing the nucleotidic sequence which encodes for the whole carbamylase. Analysis of said plasmid showed, in addition, the presence of a second incomplete ORF, placed on the other strand with respect to the carbamylase gene, which showed a homology with urease portions isolated from various microorganisms.
As ureases, like hydantoiases, are enzymes belonging to the group of amido-hydrolases, it was assumed that the incomplete ORF corresponded to that of hydantoise. This assumption was then confirmed by the enzymatic activity tests carried out on cellular extracts of cells carrying the identified gene.
In order to isolate the whole nucleotide sequence encoding hydantoinase, a screening of the gene library of the DNA of A.radiobacter digested with SacI was carried out by hybridization with an oligonucleotide synthesized on the basis of the nucleotide sequence of the incomplete ORF.
The screening led to the isolation of a clone containing the whole hydantoinase gene. The genes thus isolated were sequenced using the sequenase version Kit 2.0 sold by United State Biochemical.
For the construction of a plasmid comprising both of the isolated genes vectors selected from plasmids, cosmids and bacteriophages known in the art, can be used.
The bifunctional plasmid of E.coli and B.subtilis, pSM671 CBS 205.94 is preferably used.
This plasmid, which comprises the genes which encode for resistance to kanamycin and chloramphenicol and has replication origins operable in E.coli and B.subtilis, is characterized in that it contains a synthetic promoter capable to direct with high efficiency and without inducers, the expression of the genes put under its control.
In practice, the DNA fragments containing the genes which encode the D-hydantoinase and D-N-carbamylase enzymes are cloned into the plasmid pSM671 in the unique restriction sites EcoRI and HindIII obtaining the recombinant plasmid pSM651.
The construction can be carried out operating according to the general techniques known in the field of recombinant DNA. In order to verify whether these enzymes are expressed from B.subtilis and E.coli, cells transformed with said plasmid are cultured in a suitable culture medium. The total proteins, extracted from the cellular lysate, analyzed on polyacrylamide gel showed the presence of two proteins having a molecular weight corresponding to that of the two enzymes; these proteins represent about 10% of the total proteins. These results confirm the capacity of B.subtilis and E.coli to express said enzymes with high efficiency and without inducers.
The enzymatic system of the present invention can be obtained by culturing the strains E.coli or B.subtilis transformed with the plasmid pSM651, under aerobic conditions, in an aqueous medium containing assimilable sources of carbon and nitrogen as well as various cations, anions and, possibly, traces of vitamins, such as biotin, thiamine, or amino acids.
Assimilable carbon sources comprise carbohydrates such as glucose, hydrolized amides, molasses, sucrose or other conventional carbon sources.
Examples of nitrogen sources can be selected, for example, from mineral ammonium salts, such as ammonium nitrate, ammonium sulphate, ammonium chloride or ammonium carbonate and urea or materials containing organic or inorganic nitrogen such as peptone, yeast extract or meat extract.
The following cations and anions are equally suitable for the object of the present invention: potassium, sodium, magnesium, iron, calcium, acid phosphates, sulphates, chlorides, manganese, and nitrates.
The fermentation is carried out, under stirring, at a temperature of between 25xc2x0 and 40xc2x0 C., preferably between 30xc2x0 and 37xc2x0 C. and at a pH of between 6 and 7.5, preferably between 6.5 and 7.0.
The cells (biomass) recovered from the culture medium by means of the conventional techniques such as centrifugation or filtration are used in the conversion phase of the racemic mixtures of 5-substituted hydantoins.
Alternatively, the conversion reaction can be carried out using either the cellular extract obtained from the disintegration of the cells by sonication or French-Press, or enzymes purified or partially purified with the conventional methods, or enzymes immobilized on insoluble supports.
Numerous hydantoins substituted in position 5 can be used in the process of the present invention. Possible substituents in position 5 are selected from a linear or branched alkyl group with a number of carbon atoms of between 1 and 6, which can be mono or polysubstituted with hydroxy, carboxy, hydrosulphide or amino groups or a phenyl or benzyl group which, in turn, can contain one or more substituents in ortho, meta and para position. Examples of 5-substituted hydantoins are: D,L-5-phenylhydantoin, D,L-5-para-hydroxyphenylhydantoin, D,L-5-methylhydantoin, D,L-5-isopropylhydantoin, D,L-5-thienylhydantoin, D,L-5-para-methoxyphenylhydantoin, D,L-5-para-chloro phenylhydantoin, D,L-5-benzylhydantoin.
The conversion of the hydantoins into the corresponding D-xcex1-amino acids is carried out in a nitrogen atmosphere in a hermetically closed apparatus, at a temperature of between 20 and 60xc2x0 C., preferably between 30 and 45xc2x0 C.
The pH of the reaction medium is maintained within values of between 6 and 10 and preferably between 7 and 8.5. This regulation of the pH can be carried out, for example, by adding a base aqueous solution such as an aqueous solution of ammonia, potassium hydroxide, sodium hydroxide, sodium or potassium carbonate.
The initial concentration of the hydantoins is generally between 2% and 30% by weight. As a result of the stereospecificity of the enzymes produced from the strains of the present invention, only the D-enantiomorphs of the hydantoins are hydrolized. As hydantoins however, spontaneously racemize more or less rapidly under the operating conditions, the L-enantiomorphs are completely converted into the corresponding D-xcex1-amino acids.
The quantity of biomass which is added to the reaction mixture depends on the particular affinity of the substrate towards the enzymes. Generally a ratio by weight biomass/hydantoins of between 1/1 and 1/50 can be used.
When the conversion reaction is carried out under optimum conditions a yield of 95-98% is obtained.
The D-xcex1-amino acids prepared with the process of the present invention can be recovered from the reaction medium with the conventional methods such as ion-exchange chromatography or precipitation of the amino acid at its isoelectric point.
The plasmid pSM651 was deposited at the Bureau Voor Schimmelcultures, SK Baarn (Holland) as E.coli SMC305 where it received the deposit number CBS 203.94.